Recognition of Fibrinogen by Leukocyte Integrins

描述（由申请人提供）：白细胞整合素aM¿2 （CD11b/CD18, Mac-1）在正常的保护性炎症反应和病理性炎症中起关键作用。这种受体具有惊人的粘附和信号能力，使其成为宿主防御的首要主力。它也是许多炎症起重要作用的疾病（包括心血管疾病）的潜在治疗靶点。aM¿2的多种功能和活性源于其结合多种结构各异的配体的能力。然而，允许aM¿2表现出广泛的配体识别的机制仍然知之甚少。我们之前对aM¿2配体纤维蛋白原原型的研究为受体的ami结构域识别其配体的机制提供了初步的见解。在过去的资助期内，我们已经解决了共识的ami域识别基序，我们称之为IRM。IRM的一个关键特征是一个小核心，由许多aM¿2配体中普遍存在的碱性和疏水性氨基酸残基的特定组合组成。IRM的特征与aM¿2识别各种不相关序列的能力是一致的，从而形成aM¿2配体结合混杂的分子基础。特异性目的1是进一步表征aM¿2广泛识别特异性的机制。组合肽库和突变分析将用于阐明IRM的结构特征。质谱法将用于确定炎症相关蛋白修饰对IRM功能的影响。我们的初步研究表明，中性粒细胞分泌产物富含irm，从而可以预测它们是一类新的aM¿2配体。我们发现其中之一，人中性粒细胞cathelicidin肽LL-37，能有效结合aMb2并诱导aM¿2依赖性迁移反应。基于这一发现，我们提出LL-37和其他中性粒细胞衍生的蛋白/肽通过结合aM¿2在单核细胞/巨噬细胞上发挥其强大的免疫调节作用。特异性目的2是通过表征LL-37引起的aM - 2依赖性单核细胞反应来验证这一假设。LL-37对aM¿2信号转导和迁移功能的影响将通过表达aM¿2和缺乏aM¿2的细胞以及体内动物模型来确定。过去资助期的研究发现整合素aD¿2是一种多配体受体，其特异性与aM¿2相似，并揭示其对炎性巨噬细胞的上调抑制其迁移。特异性目的3是表征aM¿2和aD¿2的作用，这是巨噬细胞上最丰富的两种粘附性整合素，在炎症消退期间，巨噬细胞从炎症部位迁移。将在野生型和整合素缺乏小鼠中研究巨噬细胞通过引流淋巴的外排。总的来说，这些研究将导致对aM¿2控制配体识别的原理的更多理解，将为aM¿2和aD¿2的生物学提供新的见解，并可能在设计新的治疗策略中有用。

DESCRIPTION (provided by applicant): Leukocyte integrin aM¿2 (CD11b/CD18, Mac-1) plays a pivotal role in normal protective inflammatory response and pathological inflammation. This receptor has prodigious adhesive and signaling capabilities which allowed it to become the premier workhorse in host defense. It is also a potential therapeutic target in many diseases in which inflammation plays an essential role, including cardiovascular diseases. The diverse functions and activities ascribed to aM¿2 arise from its ability to bind a multitude of structurally diverse ligands. However, the mechanisms which allow aM¿2 to exhibit broad ligand recognition are still poorly understood. Our previous studies with a prototype a aM¿2 ligand fibrinogen provided initial insight into the mechanism by which the aMI-domain of the receptor recognizes its ligands. In the past funding period we have solved the consensus aMI-domain recognition motif, we termed IRM. A key feature of IRM is a small core consisting of specific combinations of basic and hydrophobic amino acid residues ubiquitous in many aM¿2 ligands. The characteristics of IRM are consistent with the capacity of aM¿2 to recognize a wide variety of unrelated sequences and, thus, form a molecular basis for aM¿2 ligand binding promiscuity. Specific Aim1 is to further characterize the mechanism underlying broad recognition specificity of aM¿2. Combinatorial peptide libraries and mutational analyses will be used to clarify the structural features of IRM. Mass spectrometry will be used to determine the effect of inflammation-associated protein modifications on the function of IRM. Our preliminary studies revealed that neutrophil secretion products are enriched in IRMs which allowed their prediction as a new class of aM¿2 ligands. We have found that one of them, human neutrophil cathelicidin peptide LL-37, effectively binds aMb2 and induces a potent aM¿2- dependent migratory response. Based on this finding we propose that LL-37 and other neutrophil-derived proteins/peptides exert their potent immunomodulatory effects by binding aM¿2 on monocyte/macrophages. Specific Aim 2 is to test this hypothesis by characterizing aM¿2-dependent monocyte responses elicited by LL-37. The effect of LL-37 on signaling and migratory functions of aM¿2 will be determined using aM¿2- expressing and aM¿2-deficient cells and in the in vivo animal model. Studies over the past funding period identified integrin aD¿2 as a multi-ligand receptor with specificity similar to that of aM¿2 and revealed that its upregulation on inflammatory macrophages inhibits their migration. Specific Aim 3 is to characterize the role of aM¿2 and aD¿2, two most abundant and adhesive integrins on macrophages, in emigration of these cells from the inflammatory site during the resolution of inflammation. The efflux of macrophages by draining lymphatics will be investigated in wild-type and integrin-deficient mice. Overall, these studies will lead to an increased understanding of the principles which govern ligand recognition by aM¿2, will give new insights into the biology of aM¿2 and aD¿2 and may be useful in the design of novel therapeutic strategies. PUBLIC HEALTH RELEVANCE: Inflammation is critically involved in the pathogenesis of many disorders, including cardiovascular disease. Integrin aM¿2 (Mac-1) is the most versatile receptor on leukocytes and mediates numerous responses of these cells during the inflammatory response. The multiplicity of functions exhibited by Mac-1 depends on its ability to bind a myriad of diverse proteins. Understanding the molecular basis for the extreme stickiness of Mac-1 and the biological significance of receptor's broad recognition could lead to new methods of treatment of disorders in which inflammation plays a role.